CN109321497B - Lysine-elongated bacillus ZJB-17009 and application thereof - Google Patents

Lysine-elongated bacillus ZJB-17009 and application thereof Download PDF

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CN109321497B
CN109321497B CN201811153847.8A CN201811153847A CN109321497B CN 109321497 B CN109321497 B CN 109321497B CN 201811153847 A CN201811153847 A CN 201811153847A CN 109321497 B CN109321497 B CN 109321497B
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柳志强
郑裕国
康雪梅
张晓健
金利群
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Zhejiang University of Technology ZJUT
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Abstract

The invention relates to a lysine bacillus (L lysinibacillus macrocrystals) ZJB-17009 and application thereof, wherein the selectivity value of L-amino acid enantiomer produced by catalyzing N-phenylacetyl-D L-amino acid reaches 99%, and the selectivity value of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid enantiomer prepared by catalyzing 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid reaches 99%.

Description

Lysine-elongated bacillus ZJB-17009 and application thereof
(I) technical field
The invention relates to a bacterial strain for producing amidohydrolase, namely lysine bacillus (L lysinibacillus macrocrystals) ZJB-17009, and application thereof in catalyzing N-phenylacetyl amino acid to prepare chiral amino acid and catalyzing N-phenylacetyl substituent of amino acid derivative to prepare chiral amino acid derivative.
(II) background of the invention
L-glufosinate-ammonium chemical name 4- [ hydroxy (methyl) phosphonyl ] -L-homoalanine, a structural analogue of L-glutamic acid, can inhibit Glutamine Synthetase (GS) activity, enable ammonia accumulation in plants, block plant light respiration action due to high-concentration ammonia accumulation, damage of chloroplast structure and vesication of matrix, and simultaneously prevent amino acid synthesis, so as to damage cell membranes and kill cells, thereby killing weeds.
The current L-glufosinate-ammonium synthesis method comprises a chemical synthesis method and a biological synthesis method, wherein the chemical method mainly comprises the following 4 methods of 1) constructing a L-glufosinate-ammonium chiral center by utilizing a chiral auxiliary reagent and carrying out chiral induction, 2) obtaining L-glufosinate-ammonium by converting natural amino acid serving as a chiral source, 3) carrying out asymmetric synthesis reaction catalyzed by a chiral catalyst, and 4) chiral resolution of a racemate, but the L-glufosinate-ammonium synthesis method by the chemical method has the advantages of multiple processes, low yield, high chiral reagent cost, large amount of three wastes and difficult treatment, and the biological method has the advantages of strict stereoselectivity, mild reaction conditions, high yield, easiness in separation and purification and the like, so that the technology for producing L-glufosinate-ammonium by the biological method has very important industrial development value.
Biological production L-glufosinate-ammonium hydrolysis of bialaphos by proteases depending on the substrate used, maintaining optical activity by stepwise synergy of phosphodiesterase I, amidase I and glutaminase hydrolysis L-3-acetamido-4- (hydroxymethylphosphono) butanamide, resolving bialaphos ethyl ester by α -chymotrypsin, deacetylation of N-acetyl-glufosinate, amidase resolution of 2-amino-4- (hydroxymethylphosphono) -butanamide, ester hydrolysis L-glufosinate-N-carboxylic anhydride, nitrile hydratase hydrolysis of glufosinate nitrile-containing substrate, transaminase catalysis of 2-carbonyl-4- (hydroxymethylphosphono) butyric acid, and the like.
The conventional chemical method for synthesizing L-glufosinate-ammonium mainly comprises 1) a chiral auxiliary agent induction method, wherein (S) -2-hydroxy-3-pinone is used as a chiral auxiliary agent for preparation, the yield is 51%, the e.e. value is 79%, D-methyl valine is used as a chiral auxiliary agent, low-temperature reaction at-78 ℃ is required, the yield is 51%, the e.e. value is 93.5%, 2) a natural amino acid chiral source method, L glutamic acid is used as a chiral source, the e.e. value is 99.4%, L-methionine is used as a chiral source, the total yield is 42.3%, the e.e. value is 93.5%, virulent iodomethane is required, 3) an asymmetric synthesis method, asymmetric catalytic hydrogenation is adopted, the catalyst dosage is large, the price of trimethylsilyl cyanide is high, an asymmetric Strecker reaction is adopted, a virulent cyanide is adopted, an asymmetric Michael addition method is adopted, the catalyst dosage is large, the product yield and the e.e. value are low, the resolution method is 86% at the highest, and the D-glufosinate-ammonium is wasted after conversion.
The important active ingredient 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid of L-glufosinate-ammonium is prepared by selectively catalyzing and resolving 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid by using amidohydrolase, and a basis is provided for realizing L-glufosinate-ammonium synthesis of an industrial route.
Chiral amino acids and derivatives thereof have an increasing role in pharmaceutical development, and the current pharmaceutical development has an increasing demand for chiral purity. The chiral amino acid production process includes chemical resolution, enzyme resolution and other processes. The enzymatic resolution has the advantages of mild condition, strong specificity, less pollution and obvious advantages compared with the chemical resolution. The amide hydrolase selectively catalyzes and resolves the N-phenylacetyl amino acid to produce the chiral amino acid, so that the chiral amino acid has wide application prospect.
Disclosure of the invention
The invention aims to provide a bacterial strain capable of producing amidohydrolase, namely lysine-elongated bacillus (L lysinibacillus macrocrystals) ZJB-17009, and application thereof in catalyzing N-phenylacetyl-D L-amino acid derivatives (2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid) to prepare important active ingredients L-glufosinate-ammonium derivatives (2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid) of L-glufosinate-ammonium or catalyzing N-phenylacetyl-D L-amino acid to prepare L-amino acid, so that a new enzyme source is provided for producing L-glufosinate-ammonium active ingredients and L-amino acid by a chemical-enzymatic method, and development of green chemical catalysis is promoted.
The technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides an amidohydrolase-producing strain, namely lysine-producing bacillus (L lysinibacillus macrocrolides) ZJB-17009, which is preserved in China center for type culture collection, and has a preservation number of CCTCC No: M2017603, a preservation date of 2017, 10 and 23, and an address of 430072, which is separated from soil by the institute of bioengineering, university of Wuhan, Zhejiang industry, and has the capability of catalyzing and synthesizing L-glufosinate.
In a second aspect, the invention provides an application of the lysine bacillus longus ZJB-17009 in preparing an important effective component L-amino acid derivative (preferably 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid) of L-glufosinate-ammonium by microbial catalytic resolution of N-phenylacetyl-D L-amino acid derivative (preferably 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid), wherein the application comprises the steps of taking wet thalli obtained by fermentation culture of the lysine bacillus longus ZJB-17009 as a catalyst, taking N-phenylacetyl-D L-amino acid derivative (preferably 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid) as a substrate, taking a buffer solution (preferably ammonia water) as a reaction medium to form a reaction system with the pH value of 8.5, carrying out a conversion reaction at the temperature of 25-55 ℃, 100-200rpm (preferably 30-40 ℃, 150rpm), after the reaction is finished, obtaining a reaction solution by separation, preferably adding the wet thalli obtained by the concentration of the wet thalli obtained by the fermentation reaction system with the concentration of the lysine bacillus longus ZJB-17009, preferably 2-5-amino acid derivative, preferably the wet thalli-200 mM concentration of the wet thalli, preferably the wet thalli obtained by the wet thalli-200 mM fermentation system, preferably the concentration of the wet thalli-200 mM fermentation system, preferably the concentration of the wet thalli-200 mM fermentation medium, preferably the wet thalli-200.
After the reaction is finished, extracting the reaction solution by using dichloromethane, adjusting the pH of a water layer to 1.0-5.0 (preferably 2.5), loading the reaction solution at the speed of 1-6.0Bv/h (preferably 4Bv/h) for ion exchange chromatography, washing the reaction solution by using deionized water, then eluting the reaction solution by using 0.2-4.5M (preferably 1M) of ammonia water at the speed of 0.5-3.0Bv/h (preferably 2Bv/h), detecting the eluent by using filter paper impregnated with 0.2% ninhydrin solution, detecting the eluent by using filter paper with the color changing to purple, wherein the eluent contains 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, collecting the eluent containing the target component, distilling the eluent under reduced pressure to paste, dissolving and recrystallizing the eluent by using methanol, taking crystals and drying the crystals to obtain the L-amino acid derivative (preferably 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid).
In the third aspect, the invention also provides an application of the lysine bacillus longus ZJB-17009 in catalyzing N-phenylacetyl-D L-amino acid to prepare L-amino acid, wherein the application uses wet thalli obtained by fermentation culture of the lysine bacillus longus ZJB-17009 as a catalyst, uses N-phenylacetyl-D L-amino acid as a substrate, uses a buffer solution (preferably ammonia water) as a reaction medium to form a conversion system with pH8.5, performs conversion reaction under the conditions of 25-55 ℃, 100 and 200rpm (preferably 30-40 ℃, 150rpm), after the reaction is finished, separates and purifies reaction liquid to obtain L-amino acid, in the conversion system, the amount of the catalyst is 20-300 g/L2 (preferably 20-60 g/L) by wet weight, the substrate is added with a final concentration of 50-500mM (preferably 100mM) by adding acetyl serine into acetyl serine, the final concentration of 50-500mM (preferably 100mM), the N-phenylacetyl-D L-amino acid is one of N-phenylacetyl-D L-alanine, N-acetyl phenylalanine, N-D6-D3, the elution solution is washed by ion exchange chromatography, the elution solution is collected by N-acetyl serine, the method after the reaction is finished, the processes of purifying, the extraction of acetyl-D-tryptophan, the extraction of acetyl serine, the extraction of acetyl serine, the extraction of acetyl-D-tryptophan, the extraction of the.
The N-phenylacetyl-D L-amino acid is prepared by the following steps of adding amino acid and NaOH into distilled water, fully stirring under an ice bath condition of 4 ℃ until the solution is colorless and transparent, dropwise adding phenylacetyl chloride, continuing to react for 2 hours under the ice bath condition of 4 ℃ after dropwise adding, stirring at normal temperature (25-30 ℃) for 5 hours until the solution is colorless and transparent, adding HCl to adjust the pH value to 1.5-5.5 (preferably 2-4), precipitating white solid, performing suction filtration and drying to obtain the N-phenylacetyl-D L-amino acid, wherein the molar ratio of the amino acid to NaOH is 1: 1-7 (preferably 1:1.5-2.5), the molar ratio of the amino acid to the phenylacetyl chloride is 1: 0.1-2 (preferably 1:0.5-1.0), the volume dosage of the distilled water is 3-10ml/g based on the mass of the amino acid, and the amino acid is one of alanine, serine, glutamic acid, tyrosine, threonine, valine, aspartic acid, methionine, leucine, isoleucine and phenylalanine.
The wet thallus obtained by fermentation culture of the lysine bacillus longus ZJB-17009 is prepared by the following method:
(1) slant culture:
inoculating lysine-long bacillus ZJB-17009 to slant culture medium with casein peptone 1-20 g/L and Na at final concentration, and culturing at 30 deg.C for 48 hr to obtain slant thallus2HPO40.5-5.0g/L，K2HPO40.5-6.0 g/L, soybean peptone 1-5 g/L, glucose 1-5 g/L1-10 g/L, agar 20 g/L, deionized water as solvent, pH 6.5-7.5, preferably slant culture medium with final concentration of casein peptone 17 g/L and Na2HPO43.0g/L，K2HPO41.5 g/L, 3 g/L of soybean peptone, 2.5 g/L5 g/L of glucose, 20 g/L of agar, deionized water as a solvent and 7.0 of pH value.
(2) Seed culture
Selecting one strain of thallus from slant thallus, inoculating to seed culture medium with final concentration of casein peptone 1-20 g/L and Na, and culturing at 30 deg.C for 24 hr to obtain seed liquid2HPO40.5-5.0g/L，K2HPO40.5-6.0 g/L, soybean peptone 1-5 g/L, glucose 1-5 g/L1-10 g/L, deionized water as solvent, pH 6.5-7.5, and final concentration of casein peptone 17 g/L, Na2HPO43.0g/L，K2HPO41.5 g/L, 3 g/L of soybean peptone, 2.5 g/L5 g/L of glucose, deionized water as solvent and pH 7.0.
(3) Fermentation culture
Inoculating the seed solution into a fermentation culture medium with the inoculation amount of 1-10% (preferably 1%) by volume concentration, performing shaking culture at 30 ℃ and 150rpm for 60h, centrifuging at 12000g for 10min, and collecting wet bacteria, wherein the final concentration of the fermentation culture medium comprises casein peptone 1-20 g/L and Na2HPO40.5-5.0g/L，K2HPO40.5-6.0 g/L percent, 1-5 g/L percent of soybean peptone, 1-5 g/L1-10 g/L percent of glucose, deionized water as solvent, pH value of 6.5-7.5, and preferably the final concentration of fermentation medium is 17 g/L percent of casein peptone, Na2HPO43.0g/L，K2HPO41.5 g/L, 3 g/L of soybean peptone, 2.5 g/L5 g/L of glucose, deionized water as solvent and pH 7.0.
The buffer solution with the pH value of 8.5 comprises the following components: a citric acid buffer (100mM, pH 4.0-6.0), a phosphoric acid buffer (100mM, pH 6.0-8.0), Tris-HCl (100mM, pH 7.0-9.0), a boric acid buffer (100mM, pH 9.0-10.5), an aqueous ammonia buffer (pH 7.0-10.0 of a substrate solution adjusted with aqueous ammonia), a Roche buffer (100mM, pH 10.5-12.0); preferably the buffer composition is: the pH of the solution was adjusted to 8.5 with ammonia.
The invention has the beneficial effects that the invention provides a bacterial strain capable of producing amidohydrolase, namely lysine bacillus (L lysinibacillus macrocrystals) ZJB-17009, and also provides a method for preparing 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, which is an important effective component of L-glufosinate by using the wet bacterial strain obtained by fermentation culture of lysine bacillus ZJB-17009 as a biocatalyst to catalyze 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid, wherein the conversion rate of 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] butyric acid reaches 49.5%, the product L-glufosinate-e reaches e.e%: 99.9%, the invention also provides a method for preparing L-amino acid by using the wet bacterial strain obtained by fermentation culture of lysine bacillus ZJB-17009 as the biocatalyst to catalyze N-phenylacetyl-D L-amino acid, the conversion rate of the product reaches 49.e% >, the chiral amino acid can reach 99.9%, the bacterial strain can be prepared by green catalysis, and the method has the advantages of mild and the application prospect of the green culture and the method has the important split reaction process.
(IV) description of the drawings:
FIG. 1 is a standard curve of OPA/NAC-glufosinate concentration under a high throughput screening method.
FIG. 2 is a liquid phase results graph of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid and 2-amino-4- [ hydroxy (methyl) phosphoryl ] -D-butyric acid under pre-column derivatization reversed phase high performance liquid chromatography.
FIG. 3 shows the result of electron microscope observation of the strain Bacillus lysinibus (L ysinibacillus macrocoides) ZJB-17009 obtained by screening.
(V) specific embodiment:
the invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
the ultrapure water is UP water, namely water with the resistivity of 18M omega cm (25 ℃), the ammonia water is an aqueous solution containing 25-28% of ammonia, the normal temperature of the ultrapure water is 25-30 ℃, ice baths in the embodiment of the invention are all 4 ℃, and the preparation method of the 2% ninhydrin solution comprises the steps of dissolving 2g of ninhydrin and 0.08g of stannous chloride in 100M L ultrapure water, stirring and filtering, and taking the filtrate for storage in a dark place.
Example 1 screening of Bacillus lysinibacillus longus (L ysinibacillus macrocrystals) ZJB-17009
1. Preliminary screening
The method comprises the steps of weighing 1g of soil sample from all parts of the country, placing the soil sample into 10m of L0.85.85% of normal saline, shaking, standing, taking supernatant into an enrichment culture medium, carrying out shaking culture at 30 ℃ and 150r/min for 2-3 days, adding 1m of L enrichment liquid into 50m of L fresh enrichment culture medium, repeating the steps for 3 times, and then carrying out separation and purification.
Bromothymol blue filter paper is selected as indicating filter paper to detect colonies capable of degrading substrates. The principle is as follows: the colonies containing the enzyme of interest, after degradation of the substrate, produce acidic by-products (phenylacetic acid, acetic acid, formic acid, benzoic acid) which cause a change in the pH locally on the indicator filter. The indicator bromothymol blue has the color change range of 5.8-7.6 (yellow-blue), so that the color of the periphery of the colony utilizing chlorine-containing organic matters is yellow, and the color of the plate around the colony which cannot be utilized is green.
Gradually diluting the enriched solution with sterile 0.85% physiological saline for 10 gradients, and selecting 10-4、10-5、10-6、10-7、10-8、10-9、10-10Spreading 0.1m L of each of five gradient dilutions on plate screening medium, incubating at 30 deg.C for 48h, spreading sterile filter paper (indicator filter paper) soaked with 10% bromothymol blue on the plate, picking the corresponding colony showing yellow on the indicator filter paper, inoculating to 96-well plate containing growth medium, incubating at 30 deg.C and 180rpm with shaking table, during which OD is detected600Drawing a growth curve, and converting when the strain grows to a logarithmic phaseInoculating 300 microliter of fresh fermentation medium, inoculating 96-well plate, and storing the residual bacteria liquid as strain in a refrigerator at 4 deg.C. And culturing the transferred 96-well plate containing the fermentation medium for 48 hours at 30 ℃ and 180rpm by a shaking table at constant temperature to ensure that the plate fully grows and produces enzyme to obtain bacterial suspension.
2. OPA/NAC high throughput screening
Dissolving a substrate 2-N-phenylacetyl-4- [ hydroxyl (methyl) phosphoryl ] -D L-butyric acid in ammonia water, adding 1ml of the bacterial suspension prepared in the step 1, adjusting the pH value of the solution to 8.5 by using the ammonia water to form a 10m L reaction system, enabling the final concentration of the substrate to be 50mM, carrying out constant temperature reaction on a shaker at 30 ℃ and 180rpm for 24 hours, correspondingly diluting the obtained conversion solution by 10 times by using ultrapure water respectively, carrying out ice bath at 4 ℃, detecting by using an OPA/NAC high-throughput screening method, and screening out a strain with a high relative fluorescence value.
OPA/NAC high throughput screening method: absorbing 40 mul of diluted ice-bath reaction liquid to a 96-hole fluorescence detection plate by using a line gun, adding the A liquid after ice-bath, oscillating for 30s in an enzyme-labeling instrument, adding 100 mul of ultrapure water, oscillating for 30s again, and carrying out gamma-fluorescence detection on the solution at the lambda positionex＝340nm；λem(455 nm) and the fluorescence intensity value obtained was combined with 2-amino-4- [ hydroxy (methyl) phosphoryl group]Comparing the standard curve of the-L-butyric acid to obtain the 2-amino-4- [ hydroxy (methyl) phosphoryl group]-L-butyric acid concentration, from which 2-amino-4- [ hydroxy (methyl) phosphoryl can be calculated]-L-butyric acid yield, obtaining the amidohydrolase biocatalytic resolution of 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl]-D L-butyric acid preparation of L-glufosinate-2-amino-4- [ hydroxy (methyl) phosphoryl]-L-conversion of butyric acid.
The solution A is prepared by dissolving N-acetyl-L-cysteine (0.448g) and o-phthalaldehyde (0.185g) in 10m L of absolute ethyl alcohol, adding a boric acid buffer solution (140mM, pH 9.5) under an ice bath condition to enable the volume to be 50m L, and storing for 3 days under the ice bath condition, wherein the final concentration of the N-acetyl-L-cysteine in the solution A is 0.313 mol/L, and the final concentration of the o-phthalaldehyde is 0.138 mol/L.
The 2-amino-4- [ hydroxy (methyl) phosphoryl group]Preparation method of standard curve of (E) -L-butyric acid comprises preparing 5 g/L g of 2-amino-4- [ hydroxy (methyl) phosphoryl group with ultrapure water]-L-butyric acid standard solution, diluted to 3 concentration gradients of 0.01-0.1 g-L (0.01, 0.02, 0.04, 0.06, 0.08, 0.1 g/L), 0.1-1.0 g/L (0.1, 0.2, 0.4, 0.6, 0.8, 1.0 g/L), 1.0-5.0 g/L (1.0, 2.0, 3.0, 4.0, 5.0 g/L), respectively sucking the standard solution with different concentration gradients from 40 mul to 96-well micropore fluorescence detection plate by a line gun, respectively adding 40 mul of the prepared A solution stored in an ice bath, respectively oscillating for 30s in an enzyme labeling instrument, then respectively adding 100 mul of ultrapure water, then oscillating for 30s, and finally carrying out the steps ofex＝340nm；λemWhen the fluorescence intensity value was measured at 455nm and the obtained fluorescence intensity value was plotted as the ordinate and the concentration of the standard substance was plotted as the abscissa, a standard curve was prepared in a total of 3 concentration ranges, and it was found that the linearity was good when the concentration of the sample was 0.01 to 0.1 g/L (see FIG. 1), R was R20.9978, showing that the method has high accuracy and applicability.
3. High performance liquid chromatography rescreening
And (3) inoculating the strains screened in the step (2) to a slant culture medium, culturing for 48h at 30 ℃, and storing in a refrigerator at 4 ℃.
The strain preserved on the slant was inoculated into a seed medium and cultured at 30 ℃ for 24 hours. Inoculating the seed liquid into a fermentation medium at an inoculation amount of 1% by volume concentration, and performing shaking culture at 30 ℃ and 150rpm for 60 h. The cells were centrifuged at 12000g for 5min to collect wet cells.
Adding a substrate 2-N-phenylacetyl-4- [ hydroxyl (methyl) phosphoryl ] -D L-butyric acid and ammonia water into wet thalli, adjusting the pH of the solution to 8.5 by using the ammonia water to form a 10m L reaction system, adjusting the final concentration of the wet thalli to 50 g/L and the final concentration of the substrate to 50mM, placing the wet thalli in a water bath shaker at 30 ℃ for conversion for 24 hours, taking 1m L conversion solution, carrying out HP L C analysis on supernate after centrifugal separation, finally obtaining a wild strain with high catalytic activity, and marking the strain as a strain ZJB-17009.
The high performance liquid chromatography (HP L C) rescreening method comprises the steps of centrifuging the conversion solution to obtain a supernatant, diluting the supernatant by 10 times with ultrapure water, taking a 200 mu l to clean 1.5m L EP tube, adding 200 mu l of derivatization reagent, reacting at 30 ℃ for 5min, adding 600 mu l of ultrapure water, uniformly mixing, filtering by a syringe filter membrane (0.22 mu m), and detecting by placing the syringe filter membrane into HP L C to obtain the concentrations of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid and 2-amino-4- [ hydroxy (methyl) phosphoryl ] -D-butyric acid in the conversion solution.
0.1g of o-phthalaldehyde and 0.12-0.12 g N-phenylacetyl-L-cysteine are used as derivatization reagents, and the derivatization reagents are dissolved in 10m L absolute ethyl alcohol and then are subjected to constant volume of 50m L by using boric acid buffer solution (100mM, pH 9.8).
The HP L C condition includes that the American Dyan U3000 high performance liquid chromatograph is provided with a fluorescence detector, and Dyan C condition18A silicon hydroxyl packed column (250mM × 4.6.6 mM), a mobile phase of ammonium acetate (50mM pH 4.7) solution containing 10% pure methanol, a column temperature controlled at 35 deg.C, and a fluorescence excitation wavelength lambdaex350nm, emission wavelength λemThe sample size was 10. mu.l at 450 nm. Under these conditions, 2-amino-4- [ hydroxy (methyl) phosphoryl group]-L-butyric acid and 2-amino-4- [ hydroxy (methyl) phosphoryl]The peak time of the-D-butyric acid is 7.800min and 9.290min (as shown in figure 2).
The enrichment medium comprises the following components: 2-N-Phenylacetyl-4- [ hydroxy (methyl) phosphoryl]-D L-butyric acid 1 g/L, glucose 5 g/L, sodium chloride 1 g/L, K2HPO4·3H2O 0.8g/L，KH2PO43.3g/L，MgSO4·7H2O0.2 g/L, deionized water as solvent, pH 7.
Plate screening medium composition: 2-N-Phenylacetyl-4- [ hydroxy (methyl) phosphoryl]-D L-butyric acid 1 g/L, glucose 5 g/L, sodium chloride 1 g/L, K2HPO4·3H2O 0.8g/L，KH2PO43.3g/L，MgSO4·7H2O0.2 g/L, agar 20 g/L, deionized water as solvent and pH 7.
The growth medium comprises 10 g/L g of sodium chloride, 10 g/L g of peptone, 5 g/L g of yeast powder and deionized water, and the composition of the slant culture medium, the seed culture medium and the fermentation culture medium is the same as that in example 3.
Example 2: identification of Strain ZJB-17009
1. Morphological identification:
the strain obtained by screening in the example 1 is inoculated on a solid culture medium, and after 24 hours of culture at 37 ℃, the strain forms a round or nearly round, soft texture, smooth and flat surface, bright light yellow colony with neat edge and luster, the diameter is 2-4mm, and the solid culture medium comprises 10 g/L of sodium chloride, 10 g/L of peptone, 5 g/L of yeast powder, 20 g/L of agar and deionized water as a solvent.
2. Physiological and biochemical identification:
94 phenotypic tests were performed on the strain ZJB-17009 using a Biolog (GEN III) automatic microbiological identification system, including 71 carbon source utilization tests and 23 chemical sensitivity tests, wherein the strain ZJB-17009 was inoculated into BUG plate medium (BIO L OG UNIVERSA L GROWTH AGAR), incubated at 33 ℃ for 2 days at constant temperature, the bacteria on the plate were washed with a sterile cotton swab and mixed with an inoculum (IF-A) to prepare a bacterial suspension, adjusted to 91% T/IF-A using a turbidimeter, the bacterial suspension was applied to each well of a biologGEN III plate using an 8-well electric liquid feeder, 100. mu. L per well, the plate was placed in a 33 ℃ incubator, and after 12h, 24h, 36h, 48h of incubation, the results were read on a Biolog reader, the strain ZJB-09 was analyzed for fingerprint metabolism using a Biolog reader, and the strain ZJB-17009 used for identifying strong carbon sources and other strains, such as the strain ZJB-1705 could not use the carbon source utilization system, and the other strains could not be identified by the Biolog-17048 system, and the chemical sensitivity test could be shown in Biolog-III system.
TABLE 1 ability of the Strain ZJB-17009 to utilize 71 carbon sources on BiologGEN III plates
Figure BDA0001818524330000091
TABLE 2 chemosensitivity of Strain ZJB-17009 to 23 chemicals on BiologGEN III plates
Figure BDA0001818524330000092
Figure BDA0001818524330000101
3. Molecular biological identification:
the method comprises the steps of using total DNA of a strain ZJB-17009 as a template, utilizing primers P1:5'-AGAGTTTGATCCTGGCTCAG-3' and P2:5'-AAGGAGGTGATCCAGCCGCA-3' to amplify 16S rDNA genes of the strain, connecting gene products with a T vector, entrusting Shanghai to amplify and sequence the 16S rDNA of the strain to obtain a 16S rDNA sequence (SEQ ID NO.1) of the strain, searching 16S rDNA gene sequences of related strains in GenBank by using B L AST on an NCBI website, and performing homology comparison.
Any nucleotide sequence obtained by substituting, deleting or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.1 in the nucleotide sequence table is within the protection scope of the present invention as long as the nucleotide sequence has homology of more than 90%.
Example 3: preparation of Wet cells
(1) Slant culture:
inoculating lysine bacillus (L ysinibacillus macrocrystals) ZJB-17009 to a slant culture medium, and culturing at 30 deg.C for 48h to obtain slant thallus;
the final concentration of the slant culture medium is 17 g/L of casein peptone and Na2HPO43.0g/L，K2HPO41.5 g/L, 3 g/L of soybean peptone, 2.5 g/L5 g/L of glucose, 20 g/L of agar, deionized water as a solvent and 7.0 of pH value.
(2) Seed culture
Selecting one strain of the thallus on the inclined plane, inoculating the strain to a seed culture medium, and culturing at 30 ℃ for 24 hours to obtain a seed solution;
the final concentration of the seed culture medium comprises 17 g/L parts of casein peptone and Na2HPO43.0g/L，K2HPO41.5 g/L, 3 g/L of soybean peptone, 2.5 g/L5 g/L of glucose, and the solvent isIonized water with pH of 7.0.
(3) Fermentation culture
Inoculating the seed solution into a fermentation culture medium in an inoculation amount of 1% of volume concentration, carrying out shaking culture at 30 ℃ and 150rpm for 60h, centrifuging at 12000g for 10min, and collecting wet thalli;
the final concentration of the fermentation medium is composed of casein peptone 17 g/L and Na2HPO43.0g/L，K2HPO41.5 g/L, 3 g/L of soybean peptone, 2.5 g/L5 g/L of glucose, deionized water as solvent and pH 7.0.
Example 4 bioconversion reaction Using 2-N-Phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid as substrate
(1) The substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid was dissolved in ammonia, 0.3g of wet cell prepared in example 3 was added, pH8.5 was adjusted with ammonia to form a 10m L reaction system, the final substrate concentration was 50mM, the system was placed in a 30 ℃ water bath shaker, and transformed at 150rpm for 24 hours, 1m L transformation solution was taken into an ep tube, and after centrifugation, the supernatant was collected and analyzed by HP L C according to example 1. the results showed that ZJB-17009 transformed the substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid to obtain the product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, with a transformation rate of 49.9% and an optical purity of 99.9%.
(2) Separating the conversion liquid containing the product 2-amino-4- [ hydroxyl (methyl) phosphoryl ] -L-butyric acid obtained by the enzyme catalytic reaction to extract the product 2-amino-4- [ hydroxyl (methyl) phosphoryl ] -L-butyric acid.
1) And (3) extraction:
adding the obtained conversion solution into a separating funnel, adding dichloromethane with the same volume at the same time, shaking, standing for 3h, discharging an organic layer from the lower end of the separating funnel, and pouring an aqueous phase layer from the upper end to obtain the aqueous phase layer which is an aqueous solution containing the product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid.
2) Product separation using anion exchange resins
① resin pretreatment
Resin 201 × 7 was soaked in warm water at 50 deg.C to fully swell the resin and remove finesSmall particles (inclined or flotation); soaking in 1.0M NaOH water solution for 3 hr, washing with deionized water to neutral, soaking in 1.0M HCl water solution for 3 hr, washing with deionized water to neutral, soaking in 1.0M NaOH water solution for 3 hr, and converting into OH-And finally washing the mixture to be neutral by deionized water for later use.
② column packing
The column is filled by a wet method (the inner diameter is 1.5cm, the height is 40cm), firstly deionized water with the height of 13cm is added into an ion exchange column, then 30m L wet resin 201 × 7 is filled into a glass cup, 50m L deionized water is added, the mixture is slowly stirred, suspended resin is poured into the ion exchange column, and the suspended resin is naturally settled, so that the endocrine of the suspended resin in the column is uniform, no obvious boundary, no air bubbles are generated, and the like.
③ and eluting
Adjusting the pH value of the aqueous solution obtained in the step 1) to 2.5, loading the sample at the column flow rate of 4.0Bv/h, taking out effluent liquid at intervals to perform liquid phase detection, when the adsorption reaches the maximum value, washing the effluent liquid with deionized water, then eluting the effluent liquid with 1.0M ammonia water at the elution speed of 2.0Bv/h, collecting eluent, and detecting the 2-amino-4- [ hydroxyl (methyl) phosphoryl group contained in the eluent at intervals]L-content of butyric acid after completion of the elution the ion exchange column was washed with deionized water and the resin was converted to OH-For the next separation.
④ purification
Distilling the eluent under reduced pressure to obtain a yellow viscous substance, adding methanol for dissolving, stirring and recrystallizing under ice bath to obtain a white solid, and filtering to obtain the product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid.
After the conversion solution 1L prepared under the reaction conditions of the step (1) is separated and purified by the method, 4.7g of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid solid is obtained, the yield of the product is 95.5%, and the optical purity is 99.9%.
Example 5 bioconversion reaction Using 2-N-Phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid as substrate
Dissolving a substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid in ammonia water, adding 0.5g of wet bacteria prepared in the method of example 3, adjusting the pH value to 8.5 by using the ammonia water to form a 10m L reaction system, wherein the final concentration of the substrate is 100mM, placing the reaction system in a water bath shaker at 30 ℃, converting the substrate for 24h at 150rpm, taking 1m L conversion solution into an ep tube, centrifuging the conversion solution, taking supernatant, performing HP L C analysis according to the method of example 1 to determine that the conversion rate is 49.9%, preparing the conversion solution 1L under the same reaction conditions, separating and purifying the conversion solution according to the method of example 4 to obtain a product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid 9.2g, wherein the yield of the product is 92.9% and the optical purity is 99.9%.
Example 6 bioconversion reaction Using 2-N-Phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid as substrate
Dissolving a substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid in ammonia water, adding 0.6g of wet bacteria prepared in the method of example 3, adjusting the pH value to 8.5 by using the ammonia water to form a 10m L reaction system, adding the substrate with the final concentration of 200mM, placing the wet bacteria in a water bath shaker at 30 ℃, converting the wet bacteria for 24h at 150rpm, taking 1m L conversion solution into an ep tube, centrifuging the conversion solution, taking supernatant, performing HP L C analysis according to the method of example 1 to determine that the conversion rate is 49.9%, preparing the conversion solution 1L under the same reaction conditions, separating and purifying the conversion solution according to the method of example 4 to obtain a product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid 18.2g, wherein the product yield is 92% and the optical purity is 99.9%.
Example 7 bioconversion reaction Using 2-N-Phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid as substrate
Dissolving a substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid in ammonia water, adding 2g of wet bacteria prepared in the method of example 3, adjusting the pH to 8.5 by using the ammonia water to form a 10m L reaction system, wherein the final concentration of the substrate is 200mM, placing the reaction system in a water bath shaker at 30 ℃, converting at 150rpm for 24h, taking 1m L conversion solution to an ep tube, centrifuging, taking supernatant, performing HP L C analysis according to the method of example 1 to determine that the conversion rate is 49.3%, preparing a conversion solution 1L under the same reaction conditions, separating and purifying according to the method of example 4 to obtain 18.4g of a product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, wherein the yield of the product is 92.9% and the optical purity is 99.9%.
Example 8 bioconversion reaction Using 2-N-Phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid as substrate
Dissolving a substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid in ammonia water, adding 0.5g of wet bacteria prepared in the method of example 3, adjusting the pH to 8.5 with the ammonia water to form a 10m L reaction system, wherein the final concentration of the substrate is 50mM, placing the reaction system in a 40 ℃ water bath shaking table, converting at 150rpm for 24h, taking 1m L conversion solution into an ep tube, centrifuging, taking supernatant, performing HP L C analysis according to the method of example 1 to determine that the conversion rate is 49.9%, preparing the conversion solution 1L under the same reaction conditions, separating and purifying according to the method of example 4 to obtain a product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid 4.6g, the product yield is 42.9%, and the optical purity is 99.9%.
Example 9 bioconversion reaction Using 2-N-Phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid as substrate
Dissolving a substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid in ammonia water, adding 2g of wet bacteria prepared in the method of example 3, adjusting the pH to 8.5 with the ammonia water to form a 10m L reaction system, wherein the final concentration of the substrate is 200mM, placing the reaction system in a 50 ℃ water bath shaking table, converting at 150rpm for 24h, taking 1m L conversion solution into an ep tube, centrifuging, taking supernatant, performing HP L C analysis according to the method of example 1 to determine that the conversion rate is 49.9%, preparing the conversion solution 1L under the same reaction conditions, separating and purifying according to the method of example 4 to obtain 19.2g of a product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, wherein the yield of the product is 96.9% and the optical purity is 99.9%.
Example 10 bioconversion reactions Using N-phenylacetyl-D L-alanine as substrate
Adding 6.0g of alanine and 4.6g of NaOH into 30ml of distilled water, fully stirring under an ice bath condition until the solution is colorless and transparent, dropwise adding 5.0g of phenylacetyl chloride, after the dropwise adding is completed, the solution is light yellow, continuing to react for 2 hours under the ice bath condition, stirring at normal temperature for reaction for 5 hours until the solution is colorless and transparent, adding HCl to adjust the pH value to about 2.0, separating out a white solid, and performing suction filtration and drying to obtain 13g of the white solid, namely the N-phenylacetyl-D L-alanine.
Dissolving a substrate N-phenylacetyl-D L-alanine in ammonia water, adding 0.6g of wet thallus prepared by the method in example 3, adjusting the pH to 8.5 by using the ammonia water to form a 10m L reaction system, wherein the final concentration of the substrate is 100mM, placing the wet thallus in a water bath shaker at 30 ℃, converting at 150rpm for 24h, taking 1m L conversion solution into an ep tube, centrifuging, taking supernatant, performing HP L C analysis according to the method in example 1 to determine that the conversion rate is 49.9%, preparing the conversion solution 1L under the same reaction conditions, separating and purifying by the method in example 4 (detecting by using filter paper soaked with 0.2% ninhydrin solution, and changing the purple black of the filter paper to indicate that the eluent contains the target substance L-alanine), and obtaining 4.2g of the product L-alanine, the yield of the product is 94.2%, and the optical purity is 99.9%.
Example 11 bioconversion reactions Using N-phenylacetyl-D L-threonine as a substrate
Adding 6.0g of threonine and 4.6g of NaOH into 30ml of distilled water, fully stirring under an ice bath condition until the solution is colorless and transparent, dropwise adding 5.0g of phenylacetyl chloride, after dropwise adding is completed, the solution is light yellow, continuing to react for 2 hours under the ice bath condition, stirring at normal temperature for reaction for 5 hours until the solution is colorless and transparent, adding HCl to adjust the pH value to about 2.0, separating out a white solid, and performing suction filtration and drying to obtain the white solid, namely 11g of N-phenylacetyl-D L-threonine.
Dissolving a substrate N-phenylacetyl-D L-threonine in ammonia water, adding 3g of wet thallus prepared by the method in example 3, adjusting the pH to 8.5 by using ammonia water to form a 100m L reaction system, wherein the substrate is added to a final concentration of 100mM, placing the wet thallus in a water bath shaker at 30 ℃, converting at 150rpm for 24h, taking 1m L conversion solution to an ep tube, centrifuging, taking supernatant, performing HP L C analysis according to the method in example 1 to determine that the conversion rate is 49.5%, preparing the conversion solution 1L under the same reaction conditions, separating and purifying by the method in example 4 (detecting by using filter paper soaked with 0.2% ninhydrin solution, and changing purple black to indicate that the eluent contains the target substance L-threonine), and obtaining 5.8g of L-threonine, the yield of 97.4% and the optical purity of 99.9%.
Example 12 bioconversion reactions Using N-phenylacetyl-D L-tyrosine as substrate
3.619g D L-tyrosine and 1.8g NaOH are added into 25ml distilled water, the mixture is fully stirred under the ice bath condition until the solution is transparent, 2.875g phenylacetyl chloride is dripped, the solution is milky after the dripping is finished, the ice bath condition is continued for reaction for 2h, the mixture is stirred at normal temperature for reaction for 5h, HCl is added for adjusting the pH value to about 2.0, a large amount of white solid is separated out, and the white solid is N-phenylacetyl-D L-tyrosine 5.0g after the suction filtration and drying.
Dissolving a substrate N-phenylacetyl-D L-tyrosine in ammonia water, adding 0.3g of wet thallus prepared by the method in example 3, adjusting the pH to 8.5 by using the ammonia water to form a 10m L reaction system, wherein the final concentration of the substrate is 100mM, placing the wet thallus in a water bath shaker at 30 ℃, converting at 150rpm for 24 hours, taking 1m L conversion solution to an ep tube, centrifuging, taking supernatant, performing HP L C analysis according to the method in example 1 to determine that the conversion rate is 49.3%, preparing the conversion solution 1L under the same reaction conditions, separating and purifying according to the method in example 4 (detecting by using filter paper soaked with 0.2% ninhydrin solution, and changing the purple black of the filter paper to indicate that the eluent contains a target substance L-tyrosine), and obtaining L-tyrosine 8.84g of a product, the yield of 97.6% of the product and the optical purity of 99.9%.
Example 13 bioconversion reactions Using N-phenylacetyl-D L-phenylalanine as substrate
Adding 3.3g D L-phenylalanine and 1.8g NaOH into 25ml of distilled water, fully stirring under ice bath condition, thoroughly brightening the solution, dropwise adding 2.875g of phenylacetyl chloride, reacting for 2h under ice bath condition after dropwise adding, stirring at normal temperature for 5h, adding HCl to adjust the pH to about 4.0, separating out a large amount of white solid, and performing suction filtration and drying to obtain 5.2g of white solid, namely N-phenylacetyl-D L-phenylalanine.
Dissolving a substrate N-phenylacetyl-D L-phenylalanine in ammonia water, adding 0.4g of wet thallus prepared by the method in example 3, adjusting the pH to 8.5 by using the ammonia water to form a 10m L reaction system, wherein the final concentration of the substrate is 100mM, placing the wet thallus in a water bath shaker at 30 ℃, converting at 150rpm for 24 hours, taking 1m L conversion solution to an ep tube, centrifuging, taking supernate, performing HP L C analysis detection according to the method in example 1 to obtain the conversion rate of 49.2%, preparing the conversion solution 1L under the same reaction conditions, separating and purifying by the method in example 4 (detection is performed by using filter paper soaked with 0.2% ninhydrin solution, and the purple black color of the filter paper shows that the eluent contains a target substance L-phenylalanine), and obtaining 7.8g of a product L-phenylalanine, wherein the yield of the product is 94.5%, and the optical purity is 99.9%.
Example 14 bioconversion reactions Using N-phenylacetyl-D L-leucine as substrate
Adding 3.92g of leucine and 3.2g of NaOH into 30ml of distilled water, fully stirring under an ice bath condition until the solution is bright, dropwise adding 6.2g of phenylacetyl chloride, reacting for 2 hours under the ice bath condition continuously, stirring at normal temperature for 5 hours, adding HCl to adjust the pH value to about 2.0, separating out a large amount of white solid, and performing suction filtration and drying to obtain the white solid, namely N-phenylacetyl-D L-leucine 7.23 g.
Dissolving a substrate N-phenylacetyl-D L-leucine in ammonia water, adding 0.6g of wet thallus prepared by the method in example 3, adjusting the pH to 8.5 by using the ammonia water to form a 10m L reaction system, wherein the final concentration of the substrate is 100mM, placing the wet thallus in a water bath shaker at 30 ℃, converting at 150rpm for 24 hours, taking 1m L conversion solution to an ep tube, centrifuging, taking supernate, performing HP L C analysis detection according to the method in example 1 to obtain the conversion rate of 49.5%, preparing the conversion solution 1L under the same reaction conditions, separating and purifying by the method in example 4 (detecting by using filter paper soaked with 0.2% ninhydrin solution, and changing the filter paper to be purple black to show that the eluent contains a target substance L-leucine), so as to obtain L-leucine 6.0g, the yield of 91.4% and the optical purity of 99.9%.
Example 15 bioconversion reaction Using N-phenylacetyl-D L-isoleucine as substrate
Adding 7.84g of isoleucine and 6.4g of NaOH into 60ml of distilled water, fully stirring under an ice bath condition, dripping 5.78g of phenylacetyl chloride into the solution to obtain a light yellow solution, continuing to react for 2 hours under the ice bath condition, stirring at normal temperature for 5 hours, adding HCl to adjust the pH value to about 2.0, enabling the solution to become colorless and transparent, separating out a small amount of white solid, and performing suction filtration and drying to obtain 7.18g of white solid, namely N-phenylacetyl-D L-isoleucine.
Dissolving a substrate N-phenylacetyl-D L-isoleucine in ammonia water, adding 0.5g of wet thallus prepared by the method in example 3, adjusting the pH to 8.5 by using the ammonia water to form a 10m L reaction system, wherein the final concentration of the substrate is 100mM, placing the wet thallus in a water bath shaker at 30 ℃, converting at 150rpm for 24h, taking 1m L conversion solution to an ep tube, centrifuging, taking supernatant, performing HP L C analysis according to the method in example 1 to determine that the conversion rate is 49.3%, preparing the conversion solution 1L under the same reaction conditions, separating and purifying by the method in example 4 (detecting by using filter paper soaked with 0.2% ninhydrin solution, and changing the color to the black to indicate that the eluent contains the target substance L-isoleucine), and obtaining a product L-isoleucine 6.2g, the product yield is 94.5%, and the optical purity is 99.9%.
Example 16 bioconversion reactions Using N-phenylacetyl-D L-serine as substrate
Adding 6.0g of serine and 4.6g of NaOH into 20ml of distilled water, fully stirring under an ice bath condition until the solution is colorless and transparent, dropwise adding 5.2g of phenylacetyl chloride, after dropwise adding is finished, the solution is light yellow, continuing to react for 2 hours under the ice bath condition, stirring at normal temperature for 5 hours until the solution is colorless and transparent, adding HCl to adjust the pH value to about 2.0, separating out a white solid, and performing suction filtration and drying to obtain the white solid, namely 12.15g of N-phenylacetyl-D L-serine.
Dissolving a substrate N-phenylacetyl-D L-serine in ammonia water, adding 0.5g of wet thallus prepared by the method in example 3, adjusting the pH value of the solution to 8.5 by using the ammonia water to form a 10m L reaction system, adding the substrate with the final concentration of 100mM, placing the wet thallus in a water bath shaker at 30 ℃, converting the wet thallus for 24 hours at 150rpm, taking 1m L conversion solution to an ep tube, centrifuging the transformation solution, taking supernatant, performing HP L C analysis according to the method in example 1 to determine that the conversion rate is 49.2 percent, preparing the conversion solution 1L under the same reaction conditions, separating and purifying the transformation solution by using filter paper impregnated with 0.2 percent ninhydrin solution (the filter paper is used for detection, and the filter paper turns purple black to indicate that the eluent contains the target substance L-serine), and obtaining 4.9g of the product L-serine, wherein the yield of the product is 93.3 percent and the optical purity is 99.9 percent.
Example 17 bioconversion reactions Using N-phenylacetyl-D L-methionine as substrate
Adding 8.94g of methionine and 7.2g of NaOH into 120ml of distilled water, fully stirring under an ice bath condition until the solution is transparent, dropwise adding 10.3g of phenylacetyl chloride, after dropwise adding, the solution is transparent, continuing to react for 2 hours under the ice bath condition, stirring at normal temperature for 5 hours, adding HCl to adjust the pH value to about 2.0, separating out a large amount of white solid, and performing reduced pressure suction filtration and drying to obtain 15.32g of N-phenylacetyl-D L-methionine.
Dissolving a substrate N-phenylacetyl-D L-methionine in ammonia water, adding 0.45g of wet thallus prepared by the method in example 3, adjusting the pH of the solution to 8.5 by using the ammonia water to form a 10m L reaction system, wherein the final concentration of the substrate is 100mM, placing the wet thallus in a water bath shaker at 30 ℃, converting at 150rpm for 24 hours, taking 1m L conversion solution to an ep tube, centrifuging, taking supernatant, performing HP L C analysis according to the method in example 1 to determine that the conversion rate is 49.6%, preparing the conversion solution 1L under the same reaction conditions, separating and purifying by using filter paper impregnated with 0.2% ninhydrin solution, and the purplish black of the filter paper indicates that the eluent contains the target substance L-methionine to obtain 7.1g of the product L-methionine, the yield of the product is 95.2%, and the optical purity is 99.9%.
Example 18 bioconversion reaction with N-phenylacetyl-D L-valine as substrate
9.36g of valine and 7.2g of NaOH are added into 60ml of distilled water, the mixture is fully stirred under the ice bath condition until the solution is transparent, 14.24g of phenylacetyl chloride is dropwise added, the solution is yellowish and slightly turbid after the dropwise addition, the mixture is continuously reacted for 2 hours under the ice bath condition, the solution is colorless and transparent after being stirred at normal temperature for 5 hours, HCl is added to adjust the pH value to about 2.0, a large amount of white solid is separated out, and the white solid, namely 18.02g of N-phenylacetyl-D L-valine, is obtained after suction filtration and drying.
Dissolving a substrate N-phenylacetyl-D L-valine in ammonia water, adding 0.2g of wet thallus prepared by the method in example 3, adjusting the pH of the solution to 8.5 by using the ammonia water to form a 10m L reaction system, adding the substrate with the final concentration of 100mM, placing the wet thallus in a water bath shaker at 30 ℃, converting the wet thallus for 24 hours at 150rpm, taking 1m L conversion solution to an ep tube, centrifuging the conversion solution, taking the supernatant, performing HP L C analysis according to the method in example 1 to determine that the conversion rate is 49.3%, preparing the conversion solution 1L under the same reaction conditions, separating and purifying the conversion solution by the method in example 4 (detecting the conversion solution by using filter paper impregnated with 0.2% ninhydrin solution, and changing the purple black color of the filter paper to indicate that the eluent contains the target substance L-valine), so as to obtain 5.6g of the product L-valine, wherein the yield of the product is 95.7% and the optical purity is 99..
Example 19 bioconversion reactions Using N-phenylacetyl-D L-glutamic acid as a substrate
Adding 5g of glutamic acid and 4.6g of NaOH into 30ml of distilled water, fully stirring under an ice bath condition until the solution is transparent, dripping 5.735g of phenylacetyl chloride, after the dripping is finished, the solution is transparent, continuing to react for 2h under the ice bath condition, stirring at normal temperature for 5h, adding HCl to adjust the pH value to about 2.0, enabling the solution to become turbid, refrigerating overnight at 4 ℃ in a refrigerator, precipitating a large amount of white solid, and performing reduced pressure suction filtration and drying to obtain 8.45g of N-phenylacetyl-D L-glutamic acid.
Dissolving a substrate N-phenylacetyl-D L-glutamic acid in ammonia water, adding 0.4g of wet thallus prepared by the method in example 3, adjusting the pH of the solution to 8.5 by using the ammonia water to form a 10m L reaction system, adding the substrate with the final concentration of 100mM, placing the solution in a water bath shaker at 30 ℃, converting at 150rpm for 24 hours, taking 1m L conversion solution to an ep tube, centrifuging, taking supernate, performing HP L C analysis detection according to the method in example 1 to obtain the conversion rate of 49.3%, preparing the conversion solution 1L under the same reaction conditions, separating and purifying by the method in example 4 (detecting by using filter paper soaked with 0.2% ninhydrin solution, and changing the purple black color of the filter paper to show that the eluent contains the target substance L-glutamic acid) to obtain 6.9g of the product L-glutamic acid, wherein the yield of the product is 93.8%, and the optical purity is 99.9%.
Sequence listing
<110> Zhejiang industrial university
<120> lysine-long bacillus ZJB-17009 and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1457
<212>DNA
<213> lysine-elongated Bacillus (L ysinibacillus macrocides)
<400>1
ccttcggcgg ctggctccaa aggttacctc accgacttcg ggtgttacaa actctcgtgg 60
tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga 120
ttactagcga ttccggcttc atgtaggcga gttgcagcct acaatccgaa ctgagaacga 180
ctttatcgga ttagctccct ctcgcgagtt ggcaaccgtt tgtatcgtcc attgtagcac 240
gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt 300
ttgtcaccgg cagtcacctt agagtgccca actaaatgat ggcaactaag atcaagggtt 360
gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc 420
acctgtcacc gttgcccccg aaggggaaac tatatctcta cagtggtcaa cgggatgtca 480
agacctggta aggttcttcg cgttgcttcg aattaaacca catgctccac cgcttgtgcg 540
ggcccccgtc aattcctttg agtttcagtc ttgcgaccgt actccccagg cggagtgctt 600
aatgcgttag ctgcagcact aaggggcgga aaccccctaa cacttagcac tcatcgttta 660
cggcgtggac taccagggta tctaatcctg tttgctcccc acgctttcgc gcctcagcgt 720
cagttacaga ccagaaagtc gccttcgcca ctggtgttcc tccaaatctc tacgcatttc 780
accgctacac ttggaattcc actttcctct tctgcactca agtcccccag tttccaatga 840
ccctccacgg ttgagccgtg ggctttcaca tcagacttaa aagaccgcct gcgcgcgctt 900
tacgcccaat aattccggac aacgcttgcc acctacgtat taccgcggct gctggcacgt 960
agttagccgt ggctttctaa taaggtaccg tcaaggtaca gccagttact actgtacttg 1020
ttcttccctt acaacagagt tttacgatcc gaaaaccttc ttcactcacg cggcgttgct 1080
ccatcaggct ttcgcccatt gtggaagatt ccctactgct gcctcccgta ggagtctggg 1140
ccgtgtctca gtcccagtgt ggccgatcac cctctcaggt cggctacgca tcgtcgcctt 1200
ggtgagccgt tacctcacca actagctaat gcgccgcggg cccatcctat agcgacagcc 1260
gaaaccgtct ttcagtcttt caccatgaag taaaagaaat tattcggtat tagccccggt 1320
ttcccggagt tatcccaaac tatagggtag gttgcccacg tgttactcac ccgtccgccg 1380
ctaacgtcaa aggagcaagc tccttttctg ttcgctcgac ttgcatgtat taggcacgcc 1440
gccagcgttc gtcctga 1457

Claims (10)

1. Lysine-long bacillus (L lysinibacillus macrocides) ZJB-17009, which is preserved in China center for type culture Collection with the preservation number of CCTCC No: M2017603, the preservation date of 2017, 10 months and 23 days, and the address of university of Wuhan, China, 430072.
2. Use of a bacillus lysinibus jb-17009 of claim 1 for catalyzing the production of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid from 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid.
3. The application as claimed in claim 2, wherein the application comprises using wet thallus obtained by fermentation culture of Bacillus lysinibacillus longus ZJB-17009 as catalyst, using 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -D L-butyric acid as substrate, using buffer solution as reaction medium to form a reaction system with pH of 8.5, carrying out conversion reaction at 25-55 ℃ and 100-200rpm, and after the reaction is finished, separating and purifying the reaction solution to obtain 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid.
4. The use according to claim 3, wherein in the reaction system, the amount of the catalyst is 10 to 200 g/L in terms of the weight of wet cells, and the final concentration of the substrate is 10 to 500mM when initially added.
5. The use according to claim 3, wherein the reaction solution is separated and purified by extracting the reaction solution with dichloromethane after the completion of the reaction, adjusting the pH of the aqueous layer to 1.0 to 5.0, subjecting the aqueous layer to ion exchange chromatography at a rate of 1 to 6.0Bv/h, washing the aqueous layer with deionized water, eluting the aqueous layer with 0.2 to 4.5M aqueous ammonia at a rate of 0.5 to 3.0Bv/h, collecting the eluate containing the objective component, distilling the eluate under reduced pressure to a paste form, dissolving the paste in methanol to recrystallize the crystals, and drying the crystals to obtain 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid.
6. Use of the bacillus lysinibus longus ZJB-17009 of claim 1 for catalyzing N-phenylacetyl-D L-amino acids to produce N-phenylacetyl-L-amino acids.
7. The application as claimed in claim 6, wherein the said application uses the wet thallus obtained by fermentation and culture of lysine-elongated bacillus ZJB-17009 as catalyst, N-phenylacetyl-D L-amino acid as substrate, buffer solution as reaction medium to form a conversion system with pH8.5, and the conversion reaction is carried out at 25-55 deg.C and 100-200rpm, after the reaction is finished, the reaction solution is separated and purified to obtain N-phenylacetyl-L-amino acid.
8. The use according to claim 7, wherein in the conversion system the catalyst is used in an amount of 20 to 300 g/L based on the weight of wet biomass and the substrate is initially added to a final concentration of 50 to 500 mM.
9. The method of claim 7, wherein the N-phenylacetyl-D L-amino acid is selected from the group consisting of N-phenylacetyl-D L-alanine, N-phenylacetyl-D L0-serine, N-phenylacetyl-D L1-glutamic acid, N-phenylacetyl-D L-tyrosine, N-phenylacetyl-D L-threonine, N-phenylacetyl-D L-valine, N-phenylacetyl-D L-aspartic acid, N-phenylacetyl-D L-methionine, N-phenylacetyl-D L-leucine, N-phenylacetyl-D L-isoleucine, and N-phenylacetyl-D L-phenylalanine.
10. The use as claimed in claim 7, wherein the wet biomass is prepared by (1) slant culture of Bacillus longus ZJB-17009 inoculated into slant culture medium with final concentration of casein peptone 17 g/L, Na, and culturing at 30 deg.C for 48 hr to obtain slant biomass2HPO43.0g/L，K2HPO41.5 g/L, 3 g/L of soybean peptone, 2.5 g/L5 g/L of glucose, 20 g/L of agar, deionized water as a solvent and a pH value of 7.0;
(2) seed culture, selecting one strain of thallus from the slant thallus, inoculating to a seed culture medium, and culturing at 30 deg.C for 24 hr to obtain a seed solution, wherein the final concentration of the seed culture medium comprises casein peptone 17 g/L and Na2HPO43.0g/L，K2HPO41.5 g/L, soybean peptone 3 g/L, glucose 2.5 g/L5 g/L, and solvent is deionizedWater, pH 7.0;
(3) the fermentation culture comprises the steps of inoculating the seed liquid into a fermentation culture medium according to the inoculation amount with the volume concentration of 1-10%, carrying out shaking culture at 30 ℃ and 150rpm for 60 hours, centrifuging for 10min at 12000g, and collecting wet thalli, wherein the final concentration of the fermentation culture medium comprises 17 g/L parts of casein peptone and Na2HPO43.0g/L，K2HPO41.5 g/L, 3 g/L of soybean peptone, 2.5 g/L5 g/L of glucose, deionized water as a solvent and a pH value of 7.0.
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